Wnt inhibitors regulate hydrogen peroxide mediated tube formation and migration of human retinal microvascular endothelial cells

Abstract

Purpose : Ocular neovascularization associated with retinopathy of prematurity is one of the primary causes of irreversible vision loss. The mechanisms involved in the occurrence of retinal neovascularization is still under investigation. One such mechanism is thought to be elevated Wnt signaling. It has been reported that Reactive Oxygen Species (ROS), such as hydrogen peroxide (H₂O₂), induces Wnt activity in pathological conditions. Several studies also report that H₂O₂ enhances angiogenesis. Thus we wanted to investigate the role of H₂O₂ and Wnt in tube formation of human retinal endothelial cells. We utilized novel small molecule Wnt inhibitors to see their effect on H₂O₂ mediated stimulates tube formation of primary human retinal microvascular endothelial cells (HRMEC) in vitro.

Methods : Quantitative PCR (qPCR) analysis of Axin2 was utilized to assess the inhibitory profile of Wnt inhibitors and activation profile of H₂O₂ upon Wnt signaling. HRMEC cells were seeded in matrigels and treated with H₂O₂, or H₂O₂ together with Wnt inhibitors or vehicle. Tube formation was examined via fluorescent microscopy. HRMEC cell migration was examined using culture inserts. HRMECs were seeded into culture-inserts and treated with H₂O₂ or H₂O₂ with inhibitors or vehicle. Migration rates were quantified by measuring the cell-free area using ImageJ.

Results : Application of H₂O₂ to the HRMEC cells lead to increased Axin2 mRNA expression. In contrast to this Wnt inhibitors attenuated H₂O₂ mediated Axin2 mRNA expression. Additionally, HRMEC cells treated with H₂O₂ in the presence of Wnt inhibitors exhibited lessened tube formation migration.

Conclusions : Application of small molecule Wnt inhibitors hampered the ability of HRMEC cells to form tubes and migrate during H₂O₂ treatment in vitro. These data suggest H202 induced, angiogenesis like, activity of HRMEC cells involves Wnt signaling. Therefore, these small molecule Wnt inhibitors may serve as useful tool to assess H₂O₂ mediated neovascularization mechanisms in vitro.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.